Author: marina02

Ultrastructural Analysis of Amoebae and Gill Lesions in Nodular Gill Disease of Rainbow Trout

In Swiss rainbow trout (Oncorhynchus mykiss) farms, Nodular Gill Disease (NGD) remains a major health challenge. In recent years, several studies have been conducted to better understand the disease. In 2023, six amoeba species were identified on the gills of NGD-affected rainbow trout, and a gill score (GS) system was developed. In 2024, Vannella mustalahtiana was detected in NGD-affected fish, with its presence increasing alongside the severity of gill lesions, suggesting a possible association with clinical NGD.

Macroscopy and histopathology of NGD-affected gills (poster presentation) was characterized by extensive proliferation of the gill epithelium, forming nodular structures at the filament tips. Affected fish experience respiratory distress, often resulting in mortality.

In previous studies, presence of amoebae was observed in native smears or using light microscopy.

The aims of this study were to (1) visualize the three-dimensional structure of involved amoebae using scanning electron microscopy (SEM), (2) examine internal organelles, and (3) characterize neighbouring gill cells using transmission electron microscopy (TEM).

Amoebae from ten different cultures, originating from six Swiss rainbow trout farms, were analyzed. The amoeba species present included Vannella sp., Ripella sp., Saccamoeba sp., and an unidentified amoeba. In addition, gill samples from eight healthy and fifteen NGD-affected rainbow trout were collected and preserved in isopropanol. Amoebae cultures were examined with SEM, while gill tissues were analyzed with TEM.

In SEM analysis, amoebae were often covered by filamentous bacteria and organic debris, complicating the visualization of their surface ultrastructure. TEM imaging successfully captured the ultrastructure of amoeba and rainbow trout gills, allowing comparisons between healthy and diseased tissues.

Preliminary observations suggest distinct ultrastructural differences between healthy and NGD-affected gills. Further analyses are ongoing to characterize amoebae at ultrastructural level.

1. Vannetti, Stefania, Institute for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University Bern,, Presenter
2. Knüsel, Ralph, fishdoc GmbH, Author
3. Kässmeyer, Sabine, Veterinary Anatomy, Vetsuisse faculty, University of Bern, Bern, Switzerland, Author
4. Gaschen, Véronique, Veterinary Anatomy, Vetsuisse faculty, University of Bern, Bern, Switzerland, Author
5. Llorda, Jaime, Microscopy Imaging Center, electron microscopy lab, Vetsuisse faculty, University of Bern, Bern, Switzerland, Author
6. Irobalieva, Rossitza, Veterinary Anatomy, Vetsuisse faculty, University of Bern, Bern, Switzerland, Author
7. Schmidt-Posthaus, Heike, Institute for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University Bern,, Author

Phylogenetic reconstruction of Paramoebidae brings into question the validity of the genus Neoparamoeba

Introduction

Throughout the literature, both Paramoeba perurans and Neoparamoeba perurans are accepted names for the causative agent of amoebic gill disease, with no consensus. Historically, the two genera were separated morphologically based on the presence or absence of microscales. However, recent studies have illustrated that morphological characteristics are unreliable, and that members of the Paramoebidae can only truly be distinguished using molecular phylogenetic approaches. Thus, this study aimed to assess the validity of the genera Paramoeba and Neoparamoeba through rigorous phylogenetic reconstruction and examine the utility of established molecular markers, to identify which are most reliable for species delimitation.

Methods

Published sequences for the mitochondrial cytochrome oxidase subunit 1 (cox1) and the nuclear small ribosomal subunit (18S) were retrieved from public databases for species across the Paramoebidae. For direct comparison, phylogenetic reconstructions were performed using both maximum likelihood (ML) and Bayesian (BI) approaches for both genes. Similarly, to verify species interrelationships, concatenated cox1 and 18S sequences were used to perform a detailed assessment of the evolutionary relationships between the Paramoebidae using both the ML and BI approaches. To assess the phylogenetic utility of cox1 and 18S, alignments were subjected to Spart explorer to delimit and compare species partitions. Sequence mutational saturation was also performed using the transition/transversion rates, along with measurements of divergence using pairwise mismatch values within and between species.

Results

ML and BI trees for both cox1 and 18S, and the concatenated genes, show distinct grouping of other Paramoebidae genera with no distinction between the Paramoeba and Neoparamoeba, indicating a single genus of Paramoeba. When assessing the utility of the markers 18S showed a higher rate of transitions compared to transversions, while cox1 showed the reverse, with higher rates of transversions, potentially indicating mutational saturation. Pairwise mismatch values between the species of Paramoeba/Neoparamoeba indicated that these were all distinct species, within a single genus. However, the values within species implied that the sequences for Paramoeba pemaquidensis may have come from separate species. This was supported by the analysis from Spart explorer.

Conclusions

Results indicate that Neoparamoeba and Paramoeba should not be considered two separate genera but should instead revert to the single genus Paramoeba. The concatenated genes show very similar relationships to the single gene markers, despite their different evolutionary signatures. To distinguish these organisms, it is vital to use a phylogenetic approach. To ensure that this is done appropriately, markers must be carefully chosen.

1. DAGEN, SARAH, SRUC, Presenter
2. Lawton, Scott, SRUC, Author
3. Bron, James, University of Stirling, Author
4. Monaghan, Sean, University of Stirling, Author
5. Annette, Boerlage, SRUC, Author

DETECTION OF EPITHELIOCYSTIS IN WILD MEDITERRANEAN FISH SPECIES: A PRELIMINARY SURVEY

Introduction

Epitheliocystis is a widespread disease affecting the gills of both wild and farmed fish globally. The disease is characterized by the development of cysts containing intracellular bacteria that are replicated within host cells. The aim of this study is to detect and assess the presence of epitheliocystis in different wild fish species from the Mediterranean Sea, exploring their etiology, interaction with the host and potential impact.

Materials and Methods

A total number of 23 fish species sampled during several scientific fishing campaigns in the Catalan coast of Mediterranean Sea between 2008 and 2020 were examined using routine histopathological techniques for presence of epitheliocystis. Preliminary results showed that epitheliocystis was present in 13 out of 23 fish species. Among these, 5 species were studied in detail: round sardinella (Sardinella aurita), European anchovy (Engraulis encrasicolus), blotched picarel (Spicara maena), red mullet (Mullus barbatus) and blue whiting (Micromesistius poutassou). From these fish species, cysts were classified into 5 typologies according to aspect, size, location in the filament and presence of cell proliferation or inflammation around the cyst.

Results

Five different types of cysts were identified: A, basophilic, mostly small (<30>100 µm, located at the base of the filament, affecting several lamellae, and showing slight proliferation and lamellar fusion.

S. aurita had the highest prevalence and intensity, and was affected by most cyst types: 1, 2 and 3. Type 3 cysts were identified in different fish species (S. aurita, S. maena and E. encrasicolus). Type 4 cysts were only identified in S. maena, and type 5 cysts were identified in M. barbatus and M. poutassou.

Conclusions

Epitheliocystis appears in multiple wild species and with different cyst morphology. Morphological differences in the cysts could be indicative of the involvement of different bacterial agents. Further studies (including molecular and TEM analyses) are in progress to confirm the etiology associated with the different cyst types.

1. IZMAYLOV-MIRGASIMOV, TIMUR, UNIVERSITAT AUTÒNOMA DE BARCELONA, Author
2. CONSTENLA, MARIA, UNIVERSITAT AUTÒNOMA DE BARCELONA, Presenter
3. KATHARIOS, PANTELIS, HELLENIC CENTRE FOR MARINE RESEARCH, Author
4. CASCARANO, MARIA CHIARA, HELLENIC CENTRE FOR MARINE RESEARCH AND UNIVERSITY OF BARI ALDO MORO, Author
5. PADRÓS, FRANCESC, UNIVERSITAT AUTÒNOMA DE BARCELONA, Author

Biochemical characteristics of digestive enzymes of fish and their intestinal cestode (Proteocephalus percae)

Introduction. Various aspects of the relationship in the parasite-host system have been intensively studied in the last few decades. Of particular interest are the mechanisms of interaction between the host and helminths of its digestive tract. Cestodes have acquired a number of features in the process of evolution that allow them to successfully implement part of their life cycle in the digestive tract of vertebrates, including fish. One of the key points are various mechanisms that allow them to successfully compete with the host for food substrates. The aim of this work is to determine the kinetic characteristics of some intestinal digestive enzymes of perch (Perca fluviatilis) and the tegument of cestodes (Proteocephalus percea).
Methodology. The fish were caught in Chany Lake (Novosibirsk Region, Russia). Cestodes were removed from perch intestines, washed in a sterile solution of 0.9% NaCl, and frozen in liquid nitrogen until subsequent analysis. Then, the perch intestinal mucosa was collected with a scalpel and also frozen in liquid nitrogen. The expression of alkaline phosphatase, aminopeptidase, and amylase was detected in P. percea. based on transcriptome analysis data. We determined the activity of the listed digestive enzymes in perch and cestodes.

Results. We have found the similar values of enzymatic activity for pH and temperature optima between perch and cestodes. But in the same time the Michaelis-Menten constants were significantly lower for aminopeptidase and amylase than the same enzymes in perch.

Conclusions. We hypothesized that the found differences Michaelis-Menten constants will allow the parasite to hydrolyze these substrates more effectively than the host.

The work was supported by the Russian Science Foundation no. 23-74-10101.

1. SOLOVYEV, MIKHAIL, ISEA SB RAS, Presenter
2. VASILENKO, VADIM, ISEA SB RAS, Author
3. VLASENKO, PAVEL, ISEA SB RAS, Author
4. KASHINSKAYA, ELENA, ISEA SB RAS, Author

Chlorella vulgaris aquafeed supplementation as microbiota-gut-brain axis modulator in Dicentrarchus labrax

Introduction: Aquaculture holds considerable potential to contribute to food security by meeting the nutritional requirements of the growing population worldwide and supporting the transition to healthier oceanic and freshwater ecosystems. It is expected to drive continued growth in global fish production, providing a decisive source of protein and nutrients for over three billion people. However, to sustainably intensify and expand aquaculture production systems, there is a need for technical innovation, appropriate governance, and regulatory frameworks across the value chain. One emerging solution is the development of sustainable aquafeed alternatives with a low carbon footprint and high nutritional and immunological boost value, such as microalgae. To optimize such alternatives, it is essential to understand how they influence physiological mechanisms such as the immune system. The intestinal microbiota, shaped by diet, acts as a functional “organ” that supports metabolism and immune balance. Disruption of this balance can lead to dysbiosis and increased disease risk. Importantly, the microbiota-gut-brain axis (MGBA) regulates numerous biological processes, including feed intake and immune responses, via interconnected pathways involving the immune and enteric nervous systems, the vagus nerve, and microbial metabolites such as short-chain fatty acids (SCFAs). Understanding how microalgae supplemented feeds influence these pathways can help improve fish health and affordability.

Methodology: To assess gut microbial communities, a whole 16S rRNA metabarcoding approach will be used via nanopore sequencing technology. Gene expression related to immune, neuroendocrine, and vagal pathways will be quantified using qPCR. ELISA will be employed to determine neurotransmitter concentrations, and Gas Chromatography–Mass Spectrometry (GC–MS) will be used to quantify SCFAs.

Results: The project will benefit: the aquaculture industry by improving fish health, thus lowering production costs; the consumer, as safe nutritious fish at affordable prices could be possible; the environment, with lower CO2 emissions and wild stock preservation; the scientific community, as new knowledge will be produced with a clear potential for the development of new sustainable functional aquafeeds based on MGBA modulation; and the general public, as chemically free fish will help to raise cultured fish acceptance.

Conclusion: A deeper understanding of the MGBA’s role in fish physiology and disease will support the development of novel therapeutic strategies focused on microbiota modulation. Ultimately, the targeted manipulation of gut microbiota can enhance resistance to pathogens, improve growth and lipid metabolism, stimulate immune responses, and contribute to overall gut health—advancing both aquaculture sustainability and fish welfare.

1. SARAIVA, MARCIA, CIIMAR, Presenter
2. Peixoto, Diogo, CIIMAR, Author
3. Hinzman, Mariana, CIIMAR, Author
4. Carvalho, Inês, CIIMAR, Author
5. Barbosa, Helena, CIIMAR, Author

THE LOCAL IMMUNE RESPONSE TRIGGERED BY CRYPTOCARYON IRRITANS INFECTION IN SHI DRUM

THE LOCAL IMMUNE RESPONSE TRIGGERED BY CRYPTOCARYON IRRITANS INFECTION IN SHI DRUM

Laura Cervera^1,2, Elena Hernández^1,2, Marta Arizcun¹, Elena Chaves-Pozo^1*

1 COMU-IEO, CSIC, Spain * elena.chaves@ieo.csic.es

2 University of Murcia, Spain

Introduction

Some parasites such as Cryptocaryon irritans constitute a great threaten for fish farms worldwide. Species diversification in aquaculture is essential to reduce diseases risks. In the Mediterranean area, shi drum (Umbrina cirrosa) is considered as a great candidate for diversification. However, up to date, very little is known about their susceptibility to pathogens or immune system. Thus, in this work we aimed to characterize the immune response of shi drum against a severe C. irritans infection.

Methodology

In a stock of shi drum breed at the facilities of Centro Oceanográfico de Murcia (COMU-IEO, CSIC), sudden mortalities, and heavy skin injuries in appeared due to a C. irritans outbreak. During the 20 days that lasted, mortalities reached 100%. Samples of skin and gills, the target site of the infection, were taken in days 1, 2, 3, 4, 5 and 8 from the start of the mortalities (dpi). Homogenates of both tissues were prepared (1 g/mL) and total levels of proteins measured by direct UV absorption. Humoral immune response at local level was evaluated by analyzing peroxidase, protease, antiprotease and bactericidal activities in skin and gills homogenates.

Results

Peroxidase activity was increased in skin and gills at the beginning of the infection recovered control levels from 4 dpi onwards. In contrast, protease activity in skin was undetectable at 1, 3 and 4 dpi while in gills was increased at 4 dpi. Antiprotease activity, in gills, decreased at 1, 5 and 8 dpi, but was increased at 1 dpi in skin. Bactericidal activity in gills was up-regulated at 2 dpi and unaltered in skin.

Conclusions

C. irritans infection in shi drum stimulates the humoral innate immune response in the target sites of infection, but not very consistent in time. The highest increases occurred until 4 dpi suggesting a rapid response to combat the infection, but not efficient enough.

Acknowledgements

This research was funded by the European Union NextGenerationEU/PRTR, MCIN/AEI10.13039/501100011033 and Fundación Séneca (ThinkInAzulprogramme: PRTR-C17.I1) and by Spanish Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (MCIN/AEI/10.13039/501100011033 grant PRE2020-093771 to L.C.).

Thanks to the technical staff of the Aquaculture facilities of the COMU-IEO, CSIC.

1. CHAVES-POZO, ELENA, COMU-IEO, CSIC, Presenter
2. Cervera, Laura, University of Murcia, Author
3. Hernández, Elena, University of Murcia, Author
4. Arizcun, Marta, COMU-IEO, CSIC, Author

Nematodes in the liver of European flounder (Platichthys flesus) from the southern Baltic Sea

Over the past decade, the presence of parasitic nematodes has been increasingly reported in numerous fish species across the Baltic Sea, with particular emphasis on the liver parasite Contracaecum spp. Anisakidae nematodes (such as Anisakis sp. and Contracaecum sp.) are zoonotic and pose a risk to human health. Surprisingly, the information related to the presence of these nematodes in European flounder (Platichthys flesus) is scarce, even though this flatfish is popular in human consumption. It is the third most commercially caught fish species in the Baltic Sea. The aims of our study were to 1) evaluate the level of European flounder infection with Anisakidae parasite in fish sampled in the Polish waters, southern Baltic Sea; and 2) compare the obtained results with available data. Liver samples were collected in February – March 2025 in two sampling areas located in the Polish marine waters: Gdańsk Basin and south of Bornholm Island. Visual inspection of 250 livers per area was conducted and revealed the presence of nematodes. Furthermore, subsamples of 50 livers per area were collected and digested in artificial gastric juice to reveal the presence of the nematodes. Identification of collected larvae was conducted based on anathomo-morphological features. Prevalence and intensity of infection were calculated and analysed in relation to the biological parameters of fish. In the Gdańsk Basin, the prevalence of nematode infection detected during visual inspection of livers was 0.8 %, and after digestion 8 % (intensity of infection 1-4); in the case of fish caught in the area south of Bornholm island these numbers were 2.8 % and 10 %, respectively, with the intensity of infection ranging 1-9. This indicates that using only visual inspection of livers leads to the underestimation of the infection rates. The presence of Anisakis sp., Contracaecum sp. and Hysterothylacium sp. in the liver of flounder was revealed. Our findings are consistent with previous research that has shown a rise in the prevalence of nematode parasites, including C. osculatum, among various fish species in the Baltic Sea over the past few years. Moreover, our results further support the idea that the increasing prevalence of Anisakidae nematodes may be linked to a rise in the abundance of grey seals, which serve as the final host in their life cycle.(The research is funded by a targeted grant from the Polish Ministry of Agriculture and Rural Development)

1. NADOLNA-AŁTYN, KATARZYNA, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Presenter
2. PIWOSZ, KATARZYNA, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Author
3. PAWLAK, JOANNA, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Author

Changes in the helminth fauna and diet of the garfish, Belone belone, from the southern Baltic Sea

The garfish Belone belone is a pelagic oceanodromous fish found in brackish and marine waters in the eastern North Atlantic Ocean and adjacent seas. In the Polish waters of the southern Baltic Sea, fish occur especially in Puck Bay. In this area, garfish is a species that is eagerly caught during its occurrence. It is observed in the coastal zone between April and June, during spawning migration. Since 2015, the importance of fish in local fishing has been observed.Knowledge related to the parasite fauna and food composition of that fish is scarce. Diet might not only be a source of nutrients but also might be an important source of fish infection.

Our studies aimed to describe the changes in the helminth fauna and diet of the garfish from the southern Baltic Sea.

The research material was collected in 2025. Standard analyses of fish were conducted, and the viscera of the fish were frozen for further analysis. The parasitological analysis of the liver (after digestion in artificial digestive juice) and the digestive tract was performed. All found parasites were collected and identified based on anatomo-morphological features. The stomach’s diet composition analysis was performed, and all found organisms were identified to the lowest possible level, depending on the level of decomposition.

In a previous study in 2015, the prevalence of parasitological infection was 24,59%. In the intestines, the most abundant were: nematode parasite Hysterothylacium sp. and tapeworm Schistocephalus solidus. The prevalence of Anisakis simplex larvae in the liver was 13,11%. The dominant element of the diet was three-spined stickleback (Gasterosteus aculeatus), present in 54,17% of fish with filled stomachs.

Recently obtained results were compared with the studies conducted in that area a decade ago to analyse the temporal changes in the parasite fauna and diet of garfish. Furthermore, it was analysed whether food elements could be the source of infection.

The project is funded by the Polish Ministry of Agriculture and Rural Development.

1. Pawlak, Joanna, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, GDYNIA, POLAND, Presenter
2. Nadolna-Ałtyn, Katarzyna, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, GDYNIA, POLAND, Author

Parasites found in salmon (Salmo salar) from the southern Baltic Sea

Surprisingly, the information related to the presence of parasites in salmon (Salmo salar) from the southern Baltic Sea is scarce, even though this fish is popular in human consumption. Salmons from Baltic rivers have been grouped into 6 Assessment Units based on management objectives, biological and genetic characteristics of the river stocks contained in a unit. However, the southern Baltic is a feeding area where mixture of the stocks was reported. Therefore, poor status of some stocks led to a fishing ban of salmon in the southern Baltic since 2022. The aim of our studies was to analyse the condition of salmon sampled in Polish marine waters between 2006-2024. Additionally, the diet composition and presence of liver and intestines parasites in samples collected between 2020-2024 were analysed. Generalized Linear Models show statistically significant decreasing linear trend in salmon condition between 2005-2017. All analysed fish have been found infected with intestinal parasite Eubothrium sp., that may significantly affect the intake of nutrients. Presence of Anisakidae nematodes Contracaecum sp. and Pseudoterranova sp. were detected in livers of salmon. The main components of the diet were sprat (Sprattus sprattus), three-spined stickleback (Gasterosteus aculeatus) and invertebrate Mysis mixta. Diet and presence of the parasites may affect the condition of the salmon. Food components are not only source of nutrients, but also way how fish become infected. (Funding: EU Multiannual Programme for Fisheries Data Collection; own research fund of National Marine Fisheries Research Institute: DOT ParaSalmon)

1. NADOLNA-AŁTYN, KATARZYNA, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Presenter
2. PAWLAK, JOANNA, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Author
3. PODOLSKA, MAGDALENA, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Author
4. LEJK, ADAM, NATIONAL MARINE FISHERIES RESEARCH INSTITUTE, Author

Trophic interactions in a sympatric pair of whitefish with different feeding habits and plerocercoids of Triaenophorus crassus (Cestoda) parasitizing their muscles: a stable isotope approach

Introduction. Parasites play an important role in aquatic ecosystems, although relatively little is known about their contribution to the complexity of the food web structure. Analysis of stable carbon and nitrogen isotopes (δ¹³C and δ¹⁵N) allows us to analyze the feeding relationships between different organisms, including parasites, in the aquatic ecosystem. Sympatric whitefish of Teletskoye Lake (Altai Republic, Russia) are represented by two forms/species, differing in a number of morphological, ecological, and biological characteristics. One of these characteristics is the feeding habits, according to which the “normal” whitefish (Coregonus lavaretus pidschian) belongs to benthivorous, and the “dwarf” whitefish (C. l. pravdinellus) to planktivorous. The feeding habits of whitefish are reflected in their parasite structure. Cestodes Triaenophorus crassus parasitizes whitefish muscles at the plerocercoid stage. The aim of this study is to determine the feeding relationships between sympatric whitefish and their muscle parasites (plerocercoids T. crassus).

Methodology. In autumn (September) we determined the content of stable isotopes ¹³C and ¹⁵N in whitefish muscles, stomach contents, plerocercoids, and food items. Whitefish were caught using gill nets with a mesh size of 20-35 mm knot-to-knot. Zooplanktonic organisms were collected using an plankton net, benthic organisms, macrophytes and detritus collected manually. The samples were oven-dried at 60°C for 24-48 hours until constant weight and then homogenized. Later, the samples (400-600 μg) were encapsulated in tin cups and analyzed using a Thermo-Finnigan Delta V Plus isotope ratio mass spectrometer coupled with a Thermo Flash 1112 elemental analyzer (Thermo Scientific, Germany).

Results. Based on the results, we determined that the parasites, based on the stable isotopes δ¹³C and δ¹⁵N, were confined to the whitefishes in which they parasitized, and that the plerocercoids from the muscles of the benthivorous always differed from the plerocercoids from the muscles of the planktivorous, which indicates close biochemical relationships in this whitefish-plerocercoid T. crassus system.

Conclusion. In addition, we can conclude that based on the isotopic signature of the parasite, it is possible to determine with a very high probability which of the whitefish (planktivorous or benthivorous) it parasitized.

This work was supported by the Russian Science Foundation no. 23-74-10101.

1. SOLOVYEV, MIKHAIL, ISEA SB RAS, Presenter
2. KASHINSKAYA, ELENA, ISEA SB RAS, Author
3. VLASENKO, PAVEL, ISEA SB RAS, Author